Mechanism of HIV-1 Env-Mediated Membrane Fusion Project Description The persistence of the worldwide AIDS pandemic underscores the need to develop new therapeutic and vaccine strategies against its causative agent, HIV-1. As the primary viral component on the HIV-1 surface responsible for cellular attachment and entry, the glycoprotein Env is an attractive target. This protein assembles as a homotrimer, with each protomer composed of two subunits, gp120 and gp41. The gp120 subunits bind cellular CD4 and chemokine receptors, events that coordinate a complex series of gp41 structural changes that culminate in fusion of viral and cellular membranes. The molecular mechanisms that guide these transitions remain largely unknown, in part due to the homotrimeric nature of Env. The presence of three identical binding sites for receptors that trigger conformational changes and for inhibitors that block this structural evolution makes it difficult to parse allosteric regulation within and between subunits. To explore the mechanisms of Env allostery, we have recently developed a novel strategy that combines the use of fusion inhibitors with functional complementation. Fusion inhibitors bind intermediate conformational states and report on the spatial and temporal exposure of different regions of the Env ectodomain. Functional complementation allows for the analysis of the structure and activity of individual protomers in the context of the trimer. These techniques, employed in combination, have proved to be a powerful new method to reveal structural features of Env during HIV-1 entry. We propose to use the strategy to ask the following questions: how are the exposure and conformational transitions of the gp41 subunits allosterically coupled to CD4- and coreceptor-binding to gp120? What impact do the first, second and third CD4 and chemokine receptor binding events have on the structure of the Env trimer and efficiency of membrane fusion? What structural features enable Env to maintain the conformational metastability of its native state? The specific aims are: (1) To characterize an open-to-closed conformational switch in native Env regulated by the trimer apex; (2) To probe receptor-controlled exposure of the gp41 ectodomain and explore the impact of multiple CD4 and chemokine receptor binding events on Env trimer activity; and (3) To decipher molecular pathways of inter- and intra- protomer allosteric communication within the Env trimer using sequence-based statistical coupling analysis. The information resulting from these experiments will provide new insights into the dynamic properties of the Env complex critical for viral entry and will facilitate new directions for therapeutic and vaccine development for the treatment and prevention of AIDS.